The role of direct heparan sulfate interactions and Ca2+ complexation in Hedgehog gradient formation in vivo

发育生物学的一个主要问题是细胞如何与邻近细胞协调它们的行为。作为发育过程中细胞通讯的中心模式，Hedgehog （Hh）形态因子在果蝇中从其来源传播到远处响应细胞和组织的直接生长和模式形成。未受损的Hh转运和梯度形成需要称为硫酸乙酰肝素（HS）的线性和高负电荷糖链的细胞外表达。然而，HS结合并固定了大多数其他细胞外可溶性蛋白，这就提出了与HS的相互作用如何促进Hh传播而不是减缓Hh传播的问题。在上一个资助期，我们通过表明与细胞表面HS的直接静电Hh相互作用启动和引导Hh运输的方式类似于在细胞核中受静电约束的dna结合蛋白运动，解决了这一悖论。DNA结合蛋白最好沿着双螺旋的轴线移动，并且两个DNA结合位点允许在糖-磷酸主干之间直接转移以提高搜索速度。结合硅、先进的体外和体内技术，我们发现Hhs也可以通过两个密集排列的正电荷位点在梯度场中的许多硫酸糖链之间直接切换，并且一个位点的正电荷减少严重损害了Hh在体外的切换及其在体内的远靶转运。因此，我们发现直接在HS链之间切换的能力代表了一个以前未知的临时编码形态原运动的决定因素，对其他HS结合蛋白具有重要意义。在这个提议中，我们的目标是利用我们在体外和体内建立的方案，扰动Hh无序n尾中带电氨基酸残基的组成和位置。我们还将研究发育组织中HS的动态表达和硫酸化，这决定了形态形成因子可接近的“微空间”。我们预计，HS硫酸盐含量增加的HS微空间在其周围组织上积累Hh，而表达低硫酸盐HS的邻近微空间将不会进入。我们将利用针对特定HS结构的单链可变片段（scFv）抗体，在发育中的翅膀和眼盘微空间中可视化HS的动态表达和硫酸化变化。我们的第三个目标是继续表征蝇系缺乏Ca2+协调Hh氨基酸。这些线路是在上一个资助期间使用我们既定的协议生成的。它们的表型显示Hh Ca2+协调对苍蝇的发育不是必需的，但对干细胞增殖和苍蝇配子体发生是特别需要的。因此，在下一个资助期，我们的目标是进一步表征Hh生物学的这一重要但知之甚少的方面。

A major question in developmental biology is how cells coordinate their behaviors with that of their neighbors. As a central model for cellular communication during development, Hedgehog (Hh) morphogens spread away from their source to direct growth and pattern formation in distant responding cells and tissues in Drosophila. Unimpaired Hh transport and gradient formation require extracellular expression of linear and highly negatively charged sugar chains called heparan sulfate (HS). However, HS binds and immobilizes most other extracellular soluble proteins, raising the question of how interactions with HS contribute to Hh spread instead of slowing it down. In the last funding period, we resolved this paradox by showing that direct electrostatic Hh interactions with cell-surface HS initiate and guide Hh transport in a similar way to electrostatically constrained DNA-binding protein movement in the nucleus. DNA-binding proteins preferably move along the axis of the double helix, and two DNA binding sites allow for direct transfer between sugar-phosphate backbones to increase search speed. Using a combination of in silico, advanced in vitro and in vivo techniques, we showed that Hhs do also switch directly between the many sugar-sulfate chains in the gradient field via two sites of densely arranged positive charge, and that positive charge reduction in one site severely impairs Hh switching in vitro and its transport to distant targets in vivo. Thus, we revealed that the ability to directly switch between HS chains represents a previously unknown determinant of temporally encoded morphogen movement with important implications for other HS-binding proteins. In this proposal, we aim to perturb the composition and position of charged amino acid residues in the disordered N-tail of Hh, using our established in vitro and in vivo protocols. We will also investigate dynamic HS expression and sulfation in developing tissues that determines accessible “microspaces” for morphogens. We expect that HS microspaces of increased HS sulfation over their surrounding tissue accumulate Hh, whereas neighboring microspaces expressing HS with lower sulfation will not be entered. We will visualize dynamic HS expression and sulfation changes in developing wing and eye disc microspaces by using single chain variable fragment (scFv) antibodies directed against defined HS structures. Our third aim is the continued characterization of fly lines made deficient in Ca2+-coordinating Hh amino acids. These lines were generated in the last funding period using our established protocol. Their phenotypes revealed that Hh Ca2+-coordination is non-essential for fly development, but is specifically required for stem cell proliferation and fly gametogenesis. In the next funding period, we therefore aim to further characterize this important yet poorly understood aspect of Hh biology.